Polyacrylamide medium for electrophoresis having improved elasticity

ABSTRACT

In a medium for electrophoresis comprising a polyacrylamide gel formed by crosslinking polymerization of an acrylamide compound and a crosslinking agent in the presence of water, and a modifier, the improvement in which the medium contains a water-soluble polymer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a medium for gel electrophoresis (hereinafterreferred to as "gel medium") used to determine the base sequence of DNA,DNA fragment and DNA derivative, and in particular provides acomposition of gel medium to improve workability of the medium.

2. Description of Prior Arts

In the method for determination of the base sequence of DNA, RNA, theirfragments, and their derivatives according to the post-label method, theoperation of slab electrophoresis using a polyacrylamide gel membrane isnow essential. The polyacrylamide membrane used for this purpose isobtainable, for instance, by a crosslinking polymerization betweenapprox. 95 parts by weight of a monomer such as acrylamide and approx. 5parts by weight of a bifunctional crosslinking agent such asN,N'-methylenebisacrylamide in an aqueous solution containing a mixtureof the monomer and the crosslinking agent and a polymerization initiator(hereinafter referred to as "gel forming solution").

Recently, as the study concerning the gene has progressed, a rapidoperation for determination of the DNA base sequence is required. Theelectrophoresis using the polyacrylamide as the electrophoresis mediummembrane is almost essential for performing the determination of DNAbase sequence, because the polyacrylamide medium gives prominently highresolution.

However, the conventional polyacrylamide gel membrane has a seriousdisadvantage that the membrane is brittle and easily breakable.Therefore, a polyacrylamide gel membrane for determination of the DNAbase sequence is generally prepared by a process in which a gel formingsolution is poured into a cell formed with two glass plates (having aclearance of 0.3 to 1 mm) to prepare a gel membrane in the cell. In thegel membrane, sample inlets must be formed so that the membrane canreceive DNA samples (e.g., Maxam-Gilbert decomposed ³² P-labelled DNA oretc.). Accordingly, a sample slot former is generally inserted into thecell after the gel forming solution is poured therein but beforegelation takes place, so that the sample slots can be formed on the gelmembrane. It is difficult to cut off the edge from a prepared membranewith a razor or the like to form the sample inlets, because the gelmembrane is very brittle and easily breakable. For this reason, theabove-stated process involving the formation of sample slots byinsertion of the sample slot former in advance of the gelation isutilized. This complicated process is a serious obstacle in producingthe polyacrylamide gel membrane in a mass scale.

The polyacrylamide gel membrane prepared as above is then keptvertically together with the glass plates, and the sample slot former isremoved. A certain amount of a sample (Maxam-Gilbert decomposed ³²P-labeled DNA or etc.) is poured into the sample slots, and the sampleis electrophoresed. The electrophoresis is continued for a certainperiod of time, one glass plate is removed carefully, and theautoradiographic process is performed on the gel membrane. Thereafter,the determination of DNA base sequence is carried out. Even in thisprocess, the gel membrane sometimes breaks when the glass plate isremoved, because the conventional gel membrane is very brittle. Thus,the brittleness of the conventional membrane is a very serious problem.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a medium forelectrophoresis which is improved in the unfavorable features such asbrittleness of the conventional polyacrylamide gel membrane fordetermination of DNA base sequence, in which the unfavorable featurearises mainly from the composition of the gel medium.

Another object of the invention is to provide a polyacrylamide gelmedium which is satisfactory in electrophoretic characteristics such asresolution and migration velocity, being inproved in brittleness, easyto handle, capable of being cut into a desired shape, and usable forproducing a gel column to be employed in the disk electrophoresis ifrequired. This means that the medium for electrophoresis provided by theinvention is remarkably improved in total characteristics including thefunction as molecular sieve, as compared with the conventional medium.

There is provided by the present invention a medium for electrophoresiscomprising a polyacrylamide gel formed by crosslinking polymerization ofan acrylamide compound and a crosslinking agent in the presence ofwater, and a modifier, which is characterized in that said mediumcontains a water-soluble polymer.

In the present invention, the aqueous gel medium can be prepared bydissolving or dispersing an acrylamide compound and a crosslinking agentin an aqueous medium and performing the crosslinking polymerizationtherebetween in the aqueous medium to form an aqueous gel medium.Hereinafter, the term "dissolving (in water)" means to include both"dissolving (in water)" and "dispersing (in water)", and the term"aqueous solution" means to include both "aqueous solution" and "aqueousdispersion", unless otherwise indicated. The aqueous medium is used toinclude an aqueous mixture of water and an organic solvent, the latterbeing optionally added.

DETAILED DESCRIPTION OF THE INVENTION

Examples of the acrylamide compound employable in the invention includeacrylamide and its homologues such as acrylamide, N-methylacrylamide ,N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, anddiacetonacrylamide, and these acrylamide homologues may be employedindependently or in combination. Acrylamide is most preferable amongthese acrylamide compounds, and said acrylamide can be also preferablyemployed in combination with one or more of other acrylamide compounds.

As the crosslinking agent used to obtain the polyacrylamide gel of theinvention, a crosslinking agent described in "Electrophoresis" 1981,2,220-228, or known as such may be employed. Examples of thecrosslinking agent include bifunctional compounds such asN,N'-methylenebisacrylamide (BIS), N,N'-propylenebisacrylamide (PBA),diacrylamide dimethylether (DAE), 1,2-diacrylamide ethyleneglycol (DEG),ethyleneureabisacrylamide (EUB), ethylene diacrylate (EDA),N,N'-diallyltartardiamide (DATD), and N,N'-bisacrylylcystamine (BAC).The crosslinking agent can be employed in an amount of approx. 1 to 30wt.%, preferably approx. 3 to 10 wt.%, based on the total weight of themonomer (i.e., acrylamide compound) and the crosslinking agent.

As the modifier, a compound containing at least one carbamoyl group canbe used. Examples of the modifier include urea and formamide. Urea ismost preferred. The modifier can be used in an amount of approx. 40 to60 wt.% based on the volume of the aqueous gel containing the monomerand crosslinking agent. In the case that urea is used as the modifier,the amount generally ranges from approx. 6 moles (approx. 360 g.) perone liter of the aqueous gel containing the monomer and crosslinkingagent to the saturation amount, preferably from approx. 7 moles (approx.420 g.) to the saturation amount.

As the water-soluble polymer, a water-soluble polymer of the additionpolymerization type or condensation polymerization type can be used.Examples of the polymer of the addition polymerization type includenon-ionic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. Examples of the polymer of thecondensation polymerization type include non-ionic water-solublepolyalkylene glycols such as polyethylene glycol and polypropyleneglycol. The water-soluble polymer of molecular weight ranging fromapprox. 10,000 to 1,000,000 is preferably used. Among thesewater-soluble polymers, polyacrylamide and polyethylene glycol arepreferable. The water-soluble polymer is used in a range of approx. 1 to50 wt.%, preferably, approx. 5 to 30 wt.%, based on the total weight ofthe monomer and crosslinking agent.

According to the present invention, the addition of a water-solublepolymer serves to impart elasticity to the gel medium, and thus modifiedgel medium is still elastic even if it is dried. Thus, the gel medium isso improved as to be almost free from the brittleness, whereby the gelmedium becomes hardly breakable. Further, the viscosity of the gelmedium can be controlled by selecting the molecular weight and amount ofthe water-soluble polymer.

A pH buffer agent can be contained in the gel medium of the invention.Any buffer agent which is able to buffer a solution to a range of pH 8.0to 9.0, preferably pH 8.2 to 8.3 can be used. Buffer agents employablein the invention are described in literatures such as "ChemistryHandbook, Fundamental Edition" compiled by The Chemical Society of Japan(Maruzen Ltd., Tokyo, 1966) pages 1312-1320; "Data for BiochemicalResearch" compiled by R. M. C. Dawson et al., second edition (Oxford atthe Clarendon Press, 1969) pages 476-508; "Biochemistry" 5, 467 (1966);and "Analytical Biochemistry" 104, pages 300-310 (1966). Examples of thebuffer agent include tris(hydroxymethyl)aminomethane (Tris),N,N-bis(2-hydroxyethyl)glycine (Bicine), Na or K salt ofN-2-hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid and Na orK salt of N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, andfurther acids, alkalis, or salts employable together with any of thesebuffer agents. Preferred examples of the buffer agent include Tris, andthe combination of boric acid and EDTA.2Na salt (pH 8.2).

In the case that the gel medium of the invention is used in the form oflayer or membrane, a gel layer or gel membrane can be prepared by aprocess in which a gel forming solution is coated by a known method onan electric insulation support having a smooth hydrophilic surface, andthe gel forming solution is crosslinked thereon to polymerization.Examples of the support include glass plate, hydrophilic polymers in theform of plate or sheet, and other polymers (e.g., polyethyleneterephthalate, polycarbonate of bisphenol A, polyvinyl chloride,vinylidene chloride-vinyl chloride copolymer, polymethylmethacrylate,polyethylene, polypropylene, cellulose acetate, and cellulose acetatepropionate) in the form of plate or sheet, a surface of which is madehydrophilic by a known surface treatment. Examples of the treatmentemployable to make the surface of these polymers hydrophilic includeknown methods such as irradiation with ultra-violet rays, glow dischargetreatment, corona discharge treatment, flame treatment, electron beamtreatment, chemical etching, or electrochemical etching.

In the case that the gel forming solution is crosslinked topolymerization on the surface of the support, the surface of the gelforming solution can be covered with a cover film, sheet, or plate. Thesame material as employable for the support can be employed as the coverfilm, sheet, and plate.

The gel medium used in the invention is formed by radicalcrosslinking-polymerization between the monomer such as acrylamide withthe bifunctional compound (crosslinking agent) in an aqueous solution inwhich the water soluble polymer is dissolved almost homogeneously. Thegel is assumed to have a structure in which the water-soluble polymer isdispersed in the three dimensional crosslinked polymer, and thewater-soluble polymer chains are entangled with the three dimensionalcrosslinked polymer chains. This structure is one of the characteristicfeatures of the gel medium of the invention.

The crosslinking polymerization can be initiated in the presence of aperoxide and/or under irradiation of ultra-violet rays. The reaction canbe further accelerated by heat and irradiation with ultra-violet rays.

As the polymerization catalyst, a known low temperature-polymerizationinitiator can be used. Examples of the initiator includeβ-dimethylaminopropionitrile (DMAPN)-ammonium peroxodisulfate,N,N,N',N'-tetramethylethylenediamine (TEMED)-ammonium peroxodisulfate,TEMED-riboflavin, and TEMED-riboflavin-hydrogen peroxide. Irradiationwith ultraviolet rays can be employed in conjunction with the initiator.

A polyol compound such as glycerol or ethylene glycol can beincorporated in the gel medium of the invention as a wetting agent. Thepolyol compound can be introduced in an amount of approx. 1 to 40 wt.%based on the volume of the aqueous gel medium. Glycerol is particularlypreferable among polyol compounds.

The present invention will be more clearly understood with reference tothe following examples, but these examples are by no means understood torestrict the invention.

EXAMPLE 1

A glass plate cell with clearance of 0.5 mm was formed using two glassplates with smooth surface and a spacer of 0.5 mm thick. The acrylamidegel composition solution (gel forming solution) set forth in Table 1 waspoured into the cell and crosslinked to polymerization to form apolyacrylamide gel membrane. One glass plate was removed after gelationwas complete, and sample slots were formed by cutting the polyacrylamidegel membrane by means of a sharp cutter.

                  TABLE 1                                                         ______________________________________                                        Gel Composition                                                                              Sample Number                                                                 1     2       3       4                                        ______________________________________                                        Gel Composition                                                               Acrylamide       11.87 g 11.87 g 11.87 g                                                                             11.87 g                                N,N'--Methylenebis-                                                                            0.63 g  0.63 g  0.63 g                                                                              0.63 g                                 acrylamide                                                                    Urea             42 g    42 g    42 g  42 g                                   Solid Polyacrylamide                                                                           None    1.25 g  2.5 g None                                   (mean M.W. 50,000)                                                            Solid Polyacrylamide                                                                           None    None    None  1.25 g                                 (mean M.W. 800,000)                                                           Tris(hydroxymethyl)-                                                                           1.08 g  1.08 g  1.08 g                                                                              1.08 g                                 aminomethane                                                                  Boric acid       0.55 g  0.55 g  0.55 g                                                                              0.55 g                                 EDTA.2Na         93 mg   93 mg   93 mg 93 mg                                  Water            (added to make 100 ml)                                       Polymerization Initiator                                                      Ammonium peroxodisulfate                                                                       1.3 ml  1.3 ml  1.3 ml                                                                              1.3 ml                                 (5 wt. % aq. sol.)                                                            N,N,N',N'--tetramethyl-                                                                        33 μl                                                                              33 μl                                                                              33 μl                                                                            33 μl                               ethylenediamine                                                               ______________________________________                                    

Remark: A combination of tris(hydroxymethyl)aminomethane, boric acid andEDTA.2Na is a buffer composition showing a buffering ability to make pH8.2.

As for Sample No. 1 (reference gel membrane), the cut end was crackedand the membrane was broken, and accordingly sample slots of sharp edgecould not be obtained. On the other hand, as for Samples Nos. 2 to 4(gel membrane according to the invention), sample slots showing sharpedge were easily obtained.

The gel membrane obtained as above was again covered with a glass plate,and the electrophoresis was performed by the conventional method toobserve the electrophoresed dye pattern.

As for Sample No. 1, serious disorder of the electrophoresed dyes wasobserved. On the other hand, as for Samples Nos. 2 to 4, no disorderswere observed, and uniform electrophoresed patterns were formed.

Accordingly, it has been confirmed that the problem in brittleness ofthe gel membrane is well improved by the present invention, as comparedwith the conventional gel membrane.

EXAMPLE 2

Gel membranes were prepared using the same gel compositions as describedin Example 1. For reference, a membrane was prepared from the samecomposition as that of Sample No. 1, except that a sample slot formerwas employed for the preparation of the sample slots (Sample No. 1-A:reference gel membrane).

The experiment for the DNA base sequence determination was performedusing these gel membranes and a sample prepared from ³² P labeled DNAdecomposed by Maxam-Gilbert method.

Sample No. 1-A and Sample Nos. 2 to 4 exhibited normal electrophoresedpatterns and the DNA base sequence were determined with no difficulty,but Sample No. 1 exhibited a disordered electrophoresed pattern.

Accordingly, it has been confirmed that the gel membrane according tothe present invention exhibits satisfactory electrophoresischaracteristics, being almost free from the brittleness.

EXAMPLE 3

A polyacrylamide gel membrane was prepared in the same manner as inExample 1 except that the solid polyacrylamide (water-soluble polymer)was replaced with polyethylene glycol (average molecular weight:200,000). Thus prepared membrane was subjected to the sameelectrophoresis. It was observed that the gel membrane exhibitedsatisfactory electrophoresis characteristics, as well as that thebrittleness of membrane was remarkably improved and the gel membrane wasfreely cut for the formation of sample slot.

Accordingly, the advantage of the invention has been confirmed.

I claim:
 1. In a medium for electrophoresis comprising a polyacrylamidegel formed by crosslinking polymerization of an acrylamide compound anda crosslinking agent in the presence of water and a modifier selectedfrom the group consisting of urea and formamide, the improvement whichcomprises said medium containing a water-soluble polymer having amolecular weight in the range of 10,000 to 1,000,000 selected from thegroup consisting of an addition type polymer and a polycondensation typepolymer, said water-soluble polymer being incorporated into the mediumin the form of a polymer in an amount of 1 to 50 wt.%, based on thetotal weight of the acrylamide compound and crosslinking agent.
 2. Themedium for electrophoresis as claimed in claim 1 wherein saidwater-soluble polymer is incorporated into the medium in the form of apolymer in an amount of 5 to 30 wt.% based on the total weight of theacrylamide compound and crosslinking agent.
 3. The medium forelectrophoresis as claimed in claim 1 or 2 wherein said water-solublepolymer is selected from the group consisting of polyvinyl alcohol,polyvinylpyrrolidone, polyacrylamide, polyethylene glycol andpolypropylene glycol.
 4. The medium for electrophoresis as claimed inclaim 1 or 2 wherein said water-soluble polymer is selected from thegroup consisting of polyacrylamide and polyethylene glycol.
 5. In amedium for electrophoresis comprising a polyacrylamide gel formed bycrosslinking polymerization of an acrylamide compound and a crosslinkingagent in the presence of water and a modifier selected from the groupconsisting of urea and formamide, the improvement which comprises saidmedium containing a water-soluble polymer having a molecular weight inthe range of 10,000 to 1,000,000 selected from the group consisting ofan addition type polymer and a polycondensation type polymer, saidwater-soluble polymer being incorporated into the medium in the form ofa polymer in an amount of 1 to 50 wt.%, based on the total weight of theacrylamide compound and crosslinking agent, said polymer beinghomogeneously dispersed in said gel.